Mouse Oocyte In Vitro Maturation, Fertilization, and Culture of Preimplantation Embryos

Mouse oocyte in vitro maturation, combined with in vitro fertilization and subsequent embryo culture, enables the generation of preimplantation embryos under defined conditions. These procedures facilitate the investigation of the biological functions and molecular mechanisms underlying oocyte maturation, fertilization, and early embryo development. However, the successful application of this approach depends on careful handling of oocytes and sperm, appropriate culture conditions, and accurate assessment of developmental progression at each stage. Here, we describe methods for isolating immature cumulus-oocyte complexes (COCs) from mouse ovaries, in vitro maturation of oocytes, in vitro fertilization, and in vitro culture of embryos to the blastocyst stage. These procedures include preparing and equilibrating media and culture dishes, isolating and selecting COCs after ovarian dissection, collecting sperm and capacitating them, performing insemination under standardized conditions, and subsequently culturing embryos for developmental assessment. Developmental progression is evaluated using morphological criteria, including the formation of male and female pronuclei, cleavage to the two-cell stage, and blastocyst formation. This protocol can be integrated into studies of oocyte maturation and maternal factor function, providing a valuable tool for basic research in mammalian reproductive biology.